Glucose-induced insulin secretion from pancreatic β-cells critically depends on the activity of ATP-sensitive K+ channels (KATP channel). We previously generated mice lacking Kir6.2, the pore subunit of the β-cell KATP channel (Kir6.2−/−), that show almost no insulin secretion in response to glucose in vitro. In this study, we compared insulin secretion by voluntary feeding (self-motivated, oral nutrient ingestion) and by forced feeding (intra-gastric nutrient injection via gavage) in wild-type (Kir6.2+/+) and Kir6.2−/− mice. Under ad libitum feeding or during voluntary feeding of standard chow, blood glucose levels and plasma insulin levels were similar in Kir6.2+/+ and Kir6.2−/− mice. By voluntary feeding of carbohydrate alone, insulin secretion was induced significantly in Kir6.2−/− mice but was markedly attenuated compared with that in Kir6.2+/+ mice. On forced feeding of standard chow or carbohydrate alone, the insulin secretory response was markedly impaired or completely absent in Kir6.2−/− mice. Pretreatment with a muscarine receptor antagonist, atropine methyl nitrate, which does not cross the blood–brain barrier, almost completely blocked insulin secretion induced by voluntary feeding of standard chow or carbohydrate in Kir6.2−/− mice. Substantial glucose-induced insulin secretion was induced in the pancreas perfusion study of Kir6.2−/− mice only in the presence of carbamylcholine. These results suggest that a KATP channel-independent mechanism mediated by the vagal nerve plays a critical role in insulin secretion in response to nutrients in vivo.